In general, the invention features cloned transgenic ungulates (e.g., bovines) in which prion protein (PrP) activity is reduced by one or more genetically engineered mutations. Because such transgenic bovines with reduced prion protein activity should be resistant to prion-related diseases such as bovine spongiform encephalopy (BSE, also known as mad cow disease), they are a safer and preferred source for agricultural and pharmaceutical products, such as human therapeutic antibodies.
Since the first case of BSE was discovered in the United Kingdom in 1986, this infectious disease has spread to other parts of the world, such as Japan. The threat of this disease has a great impact on the agricultural and pharmaceutical fields and has limited the use of cattle in these industries. Based on over a decade of research, prion protein has been identified as a substantial cause of this infectious disease. Animals having reduced prion protein activity are desirable because of their expected resistance to prion protein-related infection.
So far, transgenic mice having reduced prion protein activity have been created using conventional gene targeting strategies in homologous recombination-proficient murine embryonic stem (ES) cells. However, ES cells from ungulates such as cattle have been difficult to isolate, culture, and genetically modify.
In the generation of prion knockout sheep, fetal fibroblasts were transfected with a promoterless knockout (KO) vector because the prion protein gene is very actively expressed in fetal fibroblasts (Denning et al., Nature Biotech., 19:559-562, 2001). By using this type of knockout vector, homologously targeted clones can be selected using an appropriate drug, such as G418 or puromycin, because the promoterless drug-resistant gene (e.g., a neomycin or puromycin-resistant gene) in the knockout vector can be expressed only when it is integrated into an actively expressed gene loci. However, only one live lamb was produced by nuclear transfer of these hemizygously targeted ovine fibroblasts, and then the lamb died about 12 days after birth. Unlike murine ES cells with an unlimited life-span, somatic fibroblasts have a limited life-span, which has made somatic gene targeting difficult because of the length of time required to culture the cells under stringent drug selection to select the desired knockout cells. Furthermore, in general, the frequency of homologous recombination in somatic fibroblasts is about 10-100 fold lower than in murine ES cells. These limitations pertaining to somatic fibroblasts in addition to the low success rate of traditional nuclear transfer methods have made it difficult to produce viable livestock with a desired site-specific mutation.
Reduction of prion protein activity by gene targeting has been attempted in cattle. To our knowledge, successful generation of transgenic bovines with a mutation at the prion locus has not been reported, probably because of a lack of appropriate knockout vectors and/or nuclear transfer methods. Thus, improved knockout vectors are needed to mutate the prion locus in ungulate cells (e.g., bovine cells) with high efficiency. Additionally, improved methods for generating transgenic ungulates (e.g., bovines) from these genetically modified donor cells are desirable.